Studying Secretory Protein Synthesis in Nicotiana benthamiana Protoplasts

Transient gene expression in plant protoplasts facilitates the analysis of hybrid genes in a fast and reproducible manner. The technique is particularly powerful when studying basic conserved biochemical processes including de novo protein synthesis, modification, assembly, transport, and turnover. Unlike individual plants, protoplast suspensions can be divided into almost identical aliquots, allowing the analysis of independent variables with uncertainties restricted to minor pipetting errors/variations. Using the examples of protein secretion and ER retention, we describe the most advanced working practice of routinely preparing, electroporating, and analyzing Nicotiana benthamiana protoplasts. A single batch of electroporation-competent protoplasts permits up to 30 individual transfections. This is ideal to assess the influence of independent variables, such as point mutations, deletions or fusions, or the influence of a co-expressed effector gene in dose–response studies.